Succinic dehydrogenase and cytochrome oxidase have been assayed in permanent cell lines (HEP 1, HEP 2, and HLM), in short-term cultures of chick embryo heart cells, and in various tissues. Their activities in different cells are compared by relating them to deoxyribonucleic acid. They are very low in HEP 1, HEP 2, and HLM cells by comparison with the activities in any normal tissues examined. All the succinic dehydrogenase was shown to be located in the mitochondria of the permanent cell lines by staining with tetrazolium derivatives. Both enzymes were more active in tissues of 19-day chick embryos than in those of 11-or 14-day embryos. The increasing activities found during normal development were quickly curtailed or reversed when heart cells were grown as monolayer cultures.Digitonin-treated mitochondria produced preparations with much higher activities of cytochrome oxidase than untreated samples. Activities measured in this way were again very much lower in HEP 1, HEP 2, and HLM cells than in the normal tissues. From the derived ratio of cytochrome oxidase:succinic dehydrogenase, it was apparent that cytochrome oxidase is diminished to a greater extent than succinic dehydrogenase in both permanent cell lines and short-term cultures, by comparison with the corresponding activities in embryonic and adult tissues. The features common to the metabolism of proliferating cells in vitro and malignant cells are discussed.
THE concept that rapidly growing tumours are " nitrogen traps " has led to the suggestion that plasma and other proteins may be ingested intact by tumour cells to a much greater extent than by most normal cells (Henderson and LePage, 1959; Gey, 1956). Ingestion is generally assumed to occur by the process of pinocytosis, and increased pinocytotic activity would be consistent with increased cell surface activity of tumour cells which might be expected from their decreased adhesiveness (McCutcheon, Coman and Moore, 1948) poorer cell contacts (Mercer and Easty, 1961) and loss of contact inhibition (Abercrombie and Ambrose, 1958).The capacities of a number of normal and tumour cells to ingest fluorescent labelled proteins in vitro have been compared and the effects of population density, various media, including the addition of insulin, serum and antimetabolites, and temperature on the process have been investigated. Primary cultures were prepared by the usual techniques involving trypsinization of the chopped tissue followed by washing of the resultant cell suspensions. Aliquots, usually 2 ml., of cell suspension were added to the test tubes containing coverslips. When the cells were well attached the medium was changed and the cultures used 24 hours later. Care was taken to select replicate cultures with similar cell population densities.The fluorescent protein solution (usually 0X05 ml. of a 500 solution), together with any other reagents was added to the culture medium (2 ml.) in the test tubes. Control and treated cultures were removed from the incubator as required, washed twice with 5 ml. portions of culture medium, sealed on to a microscope slide with vaseline/paraffin wax mixture and examined by fluorescence microscopy.
Labelling of proteinsThe proteins were conjugated with fluorescein or rhodamine B isothiocyanate using 20 parts of protein to 1 of dye, by the method of Riggs et al. (1958). Unconjugated dye was removed by passing the reaction mixture through a column of
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